Peripheral nerve injury triggers the activation of the small GTPase RhoA in spinal motor and peripheral sensory neurons. C3 transferase, an exoenzyme produced by Clostridium botulinum that inactivates RhoA by ADP-ribosylation, has been successfully applied in central nervous system (CNS) lesion models to facilitate regeneration functionally and morphologically. Until now it has not been demonstrated if C3bot exerts positive effects on peripheral axon regeneration as well. In organotypic spinal cord preparations, C3bot reduced axonal growth of motoneurons, while no effect on sensory axon outgrowth from dorsal root ganglia (DRG) explants was observed. Enzymatically inactive C3E174Q was ineffective in both culture models. Spinal cord slices exhibited a significant increase in microglia/macrophages after treatment with C3bot suggesting an inflammatory component in the inhibition of axon growth. C3bot or C3E174Q were then applied into conduits implanted after transection of the sciatic nerve in rats. Functional evaluation by electrophysiology, nociception, and walking track tests did not show any significant difference between groups with active or mutant C3E174Q . Transmission electron microscopy of the regenerated nerves revealed no significant differences in the number of myelinated and unmyelinated axons 6 weeks after surgery. Compared to the CNS, the functional significance of RhoA may be limited during nerve regeneration in a growth-promoting environment.